Recombinant proteins have been manufactured conventionally by the recombinant technologies using microbes such as E. coli, etc., cultured mammalian cells, or the like Windsor, et al., Biochemistry 32, 8807-15 (1993); Bondoc, et al., Anal. Biochem. 246, 234-8 (1997); Ball, et al., Eur. Cytokine Netw. 12, 187-93 (2001).). However, in cases where cultured mammalian cells are used as the host cells, it has become enormously costly to manufacture a large scale of recombinant proteins. Furthermore, in such cases using microbes and cultured mammalian cells there exists inevitably a serious risk of contamination of pathogens such as human-infectious viruses, prions, endotoxins etc (Schillberg, et al., Naturwissenschaften 90, 145-55 (2003).).
The gene expression of recombinant proteins in the rice seeds is reported (Takaiwa, et al., Plant Biotechnology 5, 84-92 (2007).). The rice seeds are known as sites for the accumulation of proteins and a small number of different storage proteins are accumulated in their special organelles called “protein granules”. Vectors using a storage protein promoter are reported for the recombinant proteins in the rice seeds. (Yang, et al, Plant Biotechnology 5, 815-26 (2007).).
IL-10 is a cytokine produced mainly by varieties of lymphocytes such as type-2 helper T cells (Th2), regulatory T cells and the like. Although its biological activity varies widely, the foremost characteristic differentiating it from other cytokines is a suppressive activity. IL-10 suppressively controls immune function by inhibiting the production of inflammatory cytokines such as interferon-γ″ (IFN-γ) and the like (Moore, et al., Annu. Rev. Immunol. 19, 683-765 (2001).). Thus, it is expected that IL-10 may be used as a drug for treatment of inflammatory diseases, allergic diseases, and autoimmune diseases (Asadullah, et al., Pharmacol. Rev. 55, 241-69 (2003).).
A biologically inactive form of IL-10 is a monomer with a molecular weight of 18.5 kDa, and its active form is a non-covalent homodimer with a molecular weight of 37 kDa (Syto, et al., Biochemistry 37, 16943-51 (1998).). The monomer has two sets of intra-molecular disulfide bonds, and the disulfide bonds are necessary to maintain the spatial protein structure. However, the monomer cannot exist stably only by itself. On the other hand, biologically active IL-10 consisting of two monomers connected each other by inter-molecular non-covalent bonds can exist stably only by itself. In other words, the disulfide bonds in the monomer and the non-covalent bonds connecting the sub-units of two monomers are necessary to maintain the spatial protein structure and the biological activity of IL-10, and IL-10, when these either bonds are destroyed, is irreversibly denatured and loses its biological activity. IL-10 is denatured and deactivated at high temperature or in acidic conditions (pH 6 or less). There are several reports on IL-10 refolding methods (Bondoc, et al., Anal. Biochem. 246, 234-8 (1997); Ball, et al., Eur. Cytokine Netw. 12, 187-93 (2001).) in order to obtain a biologically active form of IL-10 from its denatured monomers. However, highly efficient refolding methods of IL-10 have not been developed.
Furthermore, bioactivity assay methods are used for detecting a biologically active form of IL-10. However, such methods require a technical skill and a long time. The highly efficient and conventional methods for refolding IL-10 and detecting a biologically active form of IL-10 have not been developed.
There are cases that recombinant proteins can not be extracted from transgenic plants by commonly known extraction buffer solutions containing a buffer agent, for example tris(hydroxymethyl)aminomethane (Tris) and phosphoric acid, and a surfactant, for example a non-ionic surfactant. However, practical and highly efficient extraction methods for such recombinant proteins, without losing their native structures and biological activities, have not been developed.